The present invention is generally in the area of dopamine receptors, and is specifically a gene encoding a dopamine D4 receptor, its flanking 5xe2x80x2 and 3xe2x80x2 sequences, and its derived cDNA, and methods of use thereof in screening for compounds having selective effects on the cardiovasculature and retinal tissues through interactions with the dopamine D4 receptor.
Dopamine is an important neurotransmitter in the central nervous system (CNS), where it is thought to be involved in a variety of functions including motor coordination, reproductive regulation, and generation of emotions. A distinct peripheral dopaminergic system is thought to exist, although it is less well characterized. CNS dopamine receptors have historically been divided into two major classes, D1 and D2, which can be distinguished by pharmacological, functional, and physical characteristics (Kebabian and Calne, (1979) xe2x80x9cMultiple receptors for dopaminexe2x80x9d Nature 277:93-96; Hamblin et al., (1984) xe2x80x9cInteractions of agonists with D2 dopamine receptors: evidence for a single receptor population existing in multiple agonist affinity-states in rat striatal membranesxe2x80x9d Biochem. Pharmacol. 33:877-887; Seeman et al., (1985) xe2x80x9cConversion of dopamine receptors from high to low affinity for dopaminexe2x80x9d Biochem. Pharmacol. 34:151-154; Niznik, (1987) xe2x80x9cDopamine receptors: molecular structure and functionxe2x80x9d Mol. Cell. Endocrinol. 54:1-22). Peripheral dopamine receptors have been divided into DA1 and DA2 subgroups, which share some but not all pharmacological characteristics with their CNS counterparts (Goldberg and Kohli, (1987) xe2x80x9cIdentification and characterization of dopamine receptors in the cardiovascular systemxe2x80x9d Cardiologia 32:1603-1607; Kohli et al., (1989) xe2x80x9cDopamine receptors in the stellate ganglion of the dogxe2x80x9d Eur. J. Pharmacol. 164:265-272; Brodde, (1990) xe2x80x9cPhysiology and pharmacology of cardiovascular catecholamine receptors; implications for treatment of chronic heart failurexe2x80x9d Am. Heart J. 120:1565-1572).
Molecular cloning techniques have revealed a diversity of CNS receptor subtypes in each class. All are members of the G protein-coupled receptor gene superfamily and have seven potential transmembrane (Tm) spanning domains. In contrast to most members of the G-protein coupled receptor gene family, the D2-like genes have multiple exons separated by introns both in the coding and non-coding regions. Further diversity is generated by alternative splicing.
Prototypic D2 ligand binding and signal transduction characteristics have been found for D2 (Bunzow et al., (1988) xe2x80x9cCloning and expression of a rat D2 dopamine receptor cDNAxe2x80x9d Nature 336:783-787) and D3 (Sokoloff et al., (1990) xe2x80x9cMolecular cloning and characterization of a novel dopamine receptor (D3) as a target for neurolepticsxe2x80x9d Nature 347:146-151) receptors. The recently reported human D4 receptor also has a D2-like pharmacological profile (Van Tol et al., (1991) xe2x80x9cCloning of the gene for a human dopamine D4-receptor with high-affinity for the antipsychotic clozapinexe2x80x9d Nature 350-610-614). Two distinct D1 receptors have also been cloned, called D1 (Sunahara et al., (1990) xe2x80x9cHuman dopamine D1 receptor encoded by an intronless gene on chromosome 5xe2x80x9d Nature 347:80-83; Zhou et al., (1990) xe2x80x9cCloning and expression of human and rat D1 dopamine receptorsxe2x80x9d Nature 347:76-80; Monsma et al., (1990) xe2x80x9cMolecular cloning and expression of a D1 dopamine receptor linked to adenylyl cyclase activationxe2x80x9d Proc. Natl. Acad. Sci. USA 87:6723-6727; Dearry et al., (1990) xe2x80x9cMolecular cloning and expression of the gene for a human D1 dopamine receptorxe2x80x9d Nature 347:72-76) and D5 (Sunahara et al., (1991) xe2x80x9cCloning of the gene for a human dopamine D5 receptor with higher affinity for dopamine than D1xe2x80x9d Nature 350:614-619). To date no peripheral dopamine receptor has been cloned, although it has been suggested that there is a low level of expression of D3 in kidney (Sokoloff et al., 1990).
Van Tol et al. (1991) reported the isolation of a human D4 receptor with a high affinity for the neuroleptic drug clozapine. Multiple variants of this dopamine receptor were also reported by Van Tol, et al., (1992) Nature 358, 149-154. These receptors were also the subject of PCT WO 92/10571 by State of Oregon. Although the function of these particular receptors was not identified, they are assumed to be important in binding drugs having anti-psychotic activity.
It is an object of the present invention to provide the gene, its flanking 5xe2x80x2 and 3xe2x80x2 sequences and the derived cDNA encoding another dopamine D4 receptor present in rat cells.
It is a further object of the present invention to provide methods for expression and screening of compounds binding the new dopamine D4 receptor.
It is another object of the present invention to provide a method for screening for compounds having cardiovascular activity and effects on retinal tissue which specifically bind to dopamine D4 receptors.
It is still another object of the present invention to provide a means and method for modulation of the morphology of cells expressing D4 receptors, and other dopamine receptors, by stimulation or inhibition of the receptors via exposure of the cells to specific compounds.
A gene, its 5xe2x80x2 and 3xe2x80x2 flanking sequences and the derived cDNA encoding a rat D4 dopamine receptor that is predominantly located in the cardiovascular and retinal systems is disclosed. The gene has been expressed in transfected mammalian cells and demonstrated to preferentially bind dopamine antagonists such as clozapine.
The gene and/or cDNA is useful as a probe for related D4 dopamine receptors. Expressed in appropriate cell lines, it is useful as an in vitro screen for drugs which specifically bind to the receptor. Drugs that specifically bind to the receptor are then screened using standard methodology in rats, mice or dogs, for the physiological effects. Antibodies to the protein are useful in immunocyto chemical studies, identification and isolation via flow sorting of D4 expressing cell types, and in blocking or modifying the effects of D4 agonists and/or antagonists.
Stimulation or inhibition of the D4 receptor, D2 receptor, or D3 receptor, either in cells naturally expressing the receptor or which have been transfected with cDNAs or genes encoding anyone or more of several dopamine receptors, has been demonstrated to allow modification of the cell morphology. In one example, the number and extent of branching of neurites in cells transfected with dopamine receptors is increased significantly by exposure to compounds selectively binding to the receptors.